Neonatal necrotizing enterocolitis (NEC), the acute necrosis of the small intestine, is the most common non-respiratory, life-threatening disease among infants admitted to neonatal intensive care units. Although etiologic and epidemiologic characteristics of NEC are poorly understood, two major factors closely associated with NEC are prematurity and enteral feeding. A striking observation made by several groups of scientists is that feeding breast milk reduces the risk of NEC in premature infants. Such a beneficial effect was not observed by feeding infant formula. We recently demonstrated that human milk contains protective factors that protect the intestinal epithelial barrier function from oxidative-stress induced disruption. Our preliminary studies indicate that there are at least two protective factors in human milk, one heat stable and the other heat-sensitive. On the basis of preliminary results it is hypothesized that milk-borne factors protect the intestinal mucosal barrier function. Our long-range goal is to determine the mechanism involved in breast milk-mediated protection of the gastrointestinal mucosa, and to determine the role of milk-borne mucosal protective factors in preventing neonatal gastrointestinal diseases, such as NEC. Our next step towards this goal will be to isolate two distinct milk-borne protective factors that prevent hydrogen peroxide-induced barrier disruption, and to characterize their physical and chemical properties to reveal their identity. This goal will be achieved by: 1) isolation of protective factors to homogeneity by liquid chromatography, and 2) characterization of physical and chemical properties of isolated protective factors. These studies will reveal the identity of at least two distinct mucosal protective factors in breast milk, and provide the basis for further characterization of the mechanisms involved in mucosal protection by breast milk, and their role in preventing neonatal gastrointestinal diseases